System and method for the clonal culture of epithelial cells and applications thereof

ABSTRACT

The invention relates to means and methods for evaluating and using the specific properties of a particular epithelial cell present in a biological sample. Accordingly, the invention relates to a system for the culture of epithelial cells, in which at least one clonal culture is sown with a single epithelial cell directly extracted from a biological sample of epithelial tissue. The invention also relates to a method for the culture of epithelial cells, that particularly comprises the production of clonal cultures, each being sown with a distinct and unique epithelial cell directly extracted from a biological sample of epithelial tissue, the evaluation of the cellular growth in the clonal cultures, and advantageously the analysis of the capacity of the cellular material from the clonal cultures to reconstruct a three-dimensional epithelium representative of native tissue. The invention is adapted for the parallel implementation of a very large number of clonal cultures, in particular for making large-scale tests.

The present invention relates to the field of cell biology and of tissueengineering.

More specifically, the invention proposes means and methods with whichthe specific properties of a particular epithelial cell present in abiological sample may be evaluated and utilized.

Thus, the object of the present invention is a system for cultivatingepithelial cells, in which at least one clonal culture is sown with asingle epithelial cell directly extracted from a biological sample ofepithelial tissue.

The invention further relates to a method for cultivating epithelialcells, comprising at least the steps of:

a) extracting one or more epithelial cells directly from a biologicalsample of epithelial tissue;

b) optionally, selecting at least one population and/or subpopulation ofepithelial cells from the cells extracted in step a);

c) producing a clonal culture sown with a distinct and single epithelialcell directly stemming from step a) or b); and

d) qualitatively and/or quantitatively evaluating cell growth in theclonal culture of step c).

Further, the invention is directed to applications of such a system ormethod.

In vitro systems and methods dealing with epithelial tissues such as theepidermis, find applications in fields as diverse as medical researchand clinical development, tissue engineering and toxicology.

The different pluristratified epithelial tissues (notably the epidermis,the cornea, mucous tissues . . . ) share a certain number of commonfeatures which impose constraints for designing large scale in vitrotest architectures. These general characteristics are well exemplifiedin the case of the epidermis.

The epidermis is the most superficial structure of the skin and notablyensures the barrier function thereof. In majority consisting ofkeratinocytes, it is renewed on average every 28 days. This tissuecomprises 4 layers which correspond to the 4 steps of thedifferentiation program which the keratinocytes undergo during theirmigration from the basal layer, the deepest layer, towards the stratumcorneum, the most superficial layer. The continuous physiologicalprocess of renewal of the various layers of keratinocytes is calledkeratinopoiesis.

The basal layer of the epidermis, which includes only one monocellularlayer, is the germinative compartment. It is at this layer thatproliferation of the keratinocytes is carried out. Among basalkeratinocytes, a small proportion of cells called stem cells is found,for which it is recognized that they are at the origin of the long termrenewal of the epidermis. The immediate offspring of the stem cells iscalled a population of progenitors. The latter ensure rapid short termrenewal of the epidermis.

The stem cell notion, within the human inter-follicular epidermis,therefore defines the compartment located the most upstream in thehierarchy of keratinopoiesis. These cells are notably characterized bysignificant self-renewal capacity, which progenitor cells do not have,and a fortiori the keratinocytes engaged in differentiation. Further, animportant property of the stem cells is to durably preserve thepotential for regenerating and rebuilding the epidermal tissue.

Like the other pluristratified epithelial tissues, the epidermistherefore consists of a heterogeneous assembly of cells having variabledifferentiation (or immaturity) degrees. It is generally recognized thatbasal keratinocytes represent about 10% of the whole of thekeratinocytes of the epidermis, and the compartment of the epidermalstem cells only of the order of 0.1%.

Taking into account the quantitative needs required for applying thepresent methods for functional evaluation and screening, the cellmaterial routinely collected from skin biopsies, i.e. the whole of thekeratinocytes obtained after dissociation of an epidermis sample, allowsthe building-up of cell banks with a sufficient size for use at anindustrial scale. However, the material used for building up this typeof banks corresponds to a heterogeneous assembly of cells, comprisingbasal keratinocytes having different growth capacities and supra-basalkeratinocytes in the course of differentiation and no longer having anygrowth capacity.

As regards banks of keratinocytes, for example intended for industrialproduction of kits of rebuilt epidermises, a standard method consists offreezing the cells at the end of a single multiplication step in aculture, so as to form a stock of multiple equivalent ampoules, whichare kept in liquid nitrogen. Depending on the needs, the cell ampoulesare thawed out and placed in culture in order to achieve a secondmultiplication step. At the end of the two successive multiplicationsteps, the keratinocytes have generally carried out of the order of 10doublings of population. This type of approach was moreover used foranalyzing the heterogeneity of the growth potential of humankeratinocytes (Barrandon and Green, 1987). The authors first producedprimary cultures derived from the epidermis, which they froze in liquidnitrogen. Sub-confluent secondary cultures were prepared from frozenprimary cultures. The clones obtained after cloning (third cultivationstep or “third pass”) were classified into three categories: holoclones(rapid growth), paraclones (limited growth) and meroclones (intermediatepopulation).

The keratinocytes conventionally obtained after two successivemultiplication steps may be used for producing models of rebuilttissues. Applying these systems as they are to rare cell material, suchas stem cells, is on the other hand impossible at an industrial scalewhere vast test campaigns have to be conducted.

All in all, several types of models compatible with the conducting oflarge scale in vitro test campaigns are presently available. Thebiological material used in these tests may be: 1) immortalized celllines; 2) banks of normal cell extracted from tissue biopsies andamplified in culture; 3) rebuilt three-dimensional tissues. However, forapplying such models at a large scale, it is necessary to have availablelarge amounts of cell material. For example, in the case of testsconducted on normal cells, in vitro amplified cell populations are used,which modifies certain properties thereof depending on the appliedculture parameters. Further, if the focus is on sub-populations of rarecells, such as progenitor cells or epidermal stem cells, these cells areobtained in insufficient amounts from tissue biopsies.

Gangatirkar et al. (2007) describe a method with which an epidermis maybe rebuilt from total keratinocytes or from sub-populations sorted onthe basis of distinct phenotypes. Both proposed options consist of usingcells directly after extraction from the tissue and after cell sortingon the one hand, and using the cell material after an expansion phase inculture on the other hand. However, the quantitative needs of cellmaterial for applying the described method remain unsuitable forconducting large scale test campaigns in parallel. Further, the cellmaterial used corresponds to a complex mixture of different cells, thecapacity of which for rebuilding an epidermis is used in a global way.

The heterogeneity of the cells composing the pluristratified epithelialtissues, and notably that of the keratinocytes of the epidermis, istherefore a limiting factor in the elaboration of in vitro teststrategies.

Indeed, insofar that the cells have certain characteristics which arespecific to them, they are capable of responding differently to astimulus or a stress. The same applies for pathological epithelialtissues. For example, carcinomas are very heterogeneous tumors, in whicha small proportion of tumoral stem cells represent a key target for thetreatments.

Further, when cells having distinct characteristics are mixed in aculture, the specific behavior of some of them may be modified orignored within the mixture. An “averaged” result is thus observed overthe whole of the cultivated cells. Thus, the structural and functionalproperties of an epidermis rebuilt from a heterogeneous global cellpopulation (see for example Larderet et al., 2006) are the result of thewhole of the properties of the cells put in presence of each other. Athereby rebuilt epidermis can therefore by no means reflect the specificproperties of a single cell.

Further, the applied culture conditions may more or less severely modifythe intrinsic characteristics of the cells. Indeed, it is well knownthat the fact of placing cells from an epithelial tissue in anartificial culture environment leads to modification of their nativecharacteristics. Consequently, epithelial cells used after one or moreculture steps have cell material which is no longer comparable withcells directly stemming from a tissue sample. These modifications inparticular relate to the specific phenotype of the studied cells. Forexample, it has been shown that the cultivation of human keratinocytesfreshly isolated from an epidermis perturbs the expression of adhesionmolecules and markers used for defining a phenotype of stem cells, andthis, in a variable way depending on the culture medium used (Lorenz etal. 2008).

Eventually, standard solutions which consist of working fromheterogeneous complex cell populations are not adequate for meetingpresent medical, clinical and industrial needs.

There is therefore a need for means and methods which allow access tothe individual properties of the cells stemming from pluristratifiedepithelial tissues, while being suitable for the application of largescale in vitro test campaigns, even in the case of a cell material 100to 1,000 times rarer than the general populations which are presentlyused.

The present invention for the first time meets this need by proposingculture means and methods which (i), because of their clonal nature,allow access to the individual and specific properties of cells directlystemming from pluristratified epithelial tissues, (ii) preserve theindividual potential of said cells, (iii) even when they are applied ata large scale, do not consume much cell material, which makes themsuitable for the study and exploitation at an industrial scale of theless represented cells (stem and progenitor cells), and (iv) allow cellgrowth levels to be reached which are much greater than those obtainedwith known tools.

Thus, an object of the present invention relates to a clonal culturesystem of epithelial cells optimized for evaluating and exploiting thespecific properties of a single cell, in which a culture supportcomprises at least one clonal culture sown with a single epithelial celldirectly extracted from a biological sample of epithelial tissue.

Advantageously, said culture support comprises at least two parallelclonal cultures, each of said cultures being sown with a distinct andunique epithelial cell directly extracted from said biological sample.

Preferably, such a system appears as a biochip. The clonal cultures sownin parallel are then for example microcultures. In practice, the biochipmay notably be made from culture plates comprising multiple distinctwells, for example 6, 24, 96 wells or more. Alternatively, the biochipmay have as a support, a glass plate or a plate in any other suitablematerial, on which multiple microsurfaces are created, intended toreceive the cells, for example by a surface treatment allowing the cellsto adhere and to grow thereon. Biochips made on plates may be physicallydivided into compartments, for example by means of grids, or chemically,for example following a surface treatment of the plates which preventsthe cloned cells from migrating out of their respective culturemicrosurfaces.

Another object of the present invention relates to a method for theclonal cultivation of epithelial cells, optimized for evaluating andexploiting properties specific to a single cell, comprising at least thesteps of:

a) extracting one or more epithelial cells directly from a biologicalsample of epithelial tissue;

b) optionally, selecting at least one population and/or sub-populationof epithelial cells from the cells extracted in step a);

c) producing a clonal culture sown with a distinct and single epithelialcell directly stemming from step a) or b); and

d) qualitatively and/or quantitatively evaluating cell growth in theclonal culture of step c).

Interestingly, the cells used in the method, object of the presentinvention, may be total populations of cells directly extracted fromthese tissues, and/or sub-populations thereof, sorted on the basis ofspecific characters. Thus, cell material stemming from step b) mayadvantageously correspond to one or more sub-populations enriched inepithelial progenitors and/or stem cells.

During step c) (which may be considered as a primary growth step), thethereby extracted cell preparation is used for initiating parallelclonal cultures or microcultures. The question is of sowing the cells ofinterest individually under conditions allowing their growth, forexample in separate culture wells. In practice, clonal sowings may becarried out in an automated way with technologies such as notably flowcytometry or microfluidics. Preferably, step c) comprises the productionof at least two parallel clonal cultures, each of said cultures beingsown with a distinct and single epithelial cell directly stemming fromstep a) or b).

In particular, during step d), the growth of the cloned cells isanalyzed on the basis of one or more quantitative and/or qualitativeparameters such as:

the frequency of obtained clones relatively to the number of sowncultures: clone-forming efficiency [CFE];

the proliferative potential of the clones: number of cells making upeach clone at a given culture time;

the phenotype of clones: differentiation degree of the cells making upthe clones, expression of molecular markers.

Thus, against every expectation, the inventors were able to observe thatthe growth potential of cells cultivated according to the clonal culturemethod of the present invention is higher than that of cells cultivatedaccording to standard procedures (see Example B hereafter).

In a particular embodiment, the clonal culture method of the inventioncomprises at least the steps of:

a) extracting one or more epithelial cells in the form of monocellularsuspension(s), directly from a biological sample of epithelial tissue;

b) selecting at least one population and/or sub-population of epithelialcells from the cells extracted in step a);

c) producing a clonal culture sown with a distinct and single epithelialcell stemming from step b); and

d) qualitatively and/or quantitatively evaluating cell growth in theclonal culture of step c).

According to another embodiment, the method, object of the presentinvention, further comprises step e) consisting of amplifying the cellpopulation of the clonal culture of step c), or its offspring, by one ormore successive sub-cultures.

The question here is to produce from cell clones obtained at the end ofthe primary growth step c), long term parallel independent cell culturesvia successive sub-cultures amplified for several weeks. Depending onthe needs, the amplification may be conducted over periods for exampleranging from 2 to about 8 weeks, or even longer (cf. Exemplaryembodiment No. 1 C.2, below). With these independent cultures, it ispossible to obtain a large amount of cells which may be frozen andstored in the form of one or more banks of cells, for subsequent use.

Clonal cell banks which may thereby be obtained also represent an objectof the present invention. These banks are distinguished from existingbanks by the fact that they integrate clonal cell cultures which givethem highly specific structural and functional properties.

In another additional or alternative embodiment, the method according tothe invention further comprises step f) consisting of evaluating thetissue reconstruction potential of the cell population of the clonalculture of step c) or of its offspring. More specifically, step f)preferably consists of using the cell population of the clonal cultureof step c), or its offspring, in order to rebuild a three-dimensionaltissue, so as to evaluate its tissue reconstruction potential.

In practice, for carrying out this step, cells from primary clonalcultures or microcultures may be detached from their culture support,and they may then be used individually for each clone of interest, inorder to produce a three-dimensional organotypic culture model (forexample, a rebuilt epithelium, epidermis or skin).

Three-dimensional tissues rebuilt from clonal cultures, which may beobtained at the end of step f) of the method according to the invention,are part of the objects of the present invention. These tissues areproduced according to a novel three-dimensional organotypic model sincethe structural and functional characteristics of the tissues, object ofthe invention, are quite specific insofar that they result from theproperties of a single cell. Such tissues are notably selected fromvarious epithelial tissues, the skin, the epidermis.

Further, biochips comprising at least one tissue, as described above,form another object of the invention. These biochips may for example beformed from microcultures made within a three-dimensional gel made froma biomaterial compatible with cell growth. Systems based on multiplerebuilt three-dimensional microtissues may also be contemplated, eachbeing generated independently, directly within the biochip, without anyprior culture step.

In another additional or alternative embodiment, the method according tothe invention further comprises step g) consisting of evaluating thelong term expansion potential of the cell population of the clonalculture of step c). More specifically, step g) preferably consists ofsub-cultivating the cell population of the clonal culture of step c),under conditions promoting cell expansion until exhaustion of theexpansion potential, so as to evaluate the long term expansion potentialof said cell population.

For example, cells stemming from primary clonal cultures ormicrocultures may be detached from their culture support, and then besub-cultivated under conditions promoting their multiplication, untilexhaustion of their multiplication potential.

In another additional or alternative embodiment, the method according tothe invention further comprises step h) consisting of evaluating theclone-forming potential of the offspring of the cell population of theclonal culture of step c). More specifically, step h) preferablyconsists of evaluating the clone-forming potential of the offspring ofthe cell population of the clonal culture of step c), by means of aquantitative test of clonogenicity in which strictly clonal secondarycultures and/or low density cultures allowing growth of individualizedcolonies are made.

For this, for example, the cells forming each clone may be detached fromtheir culture support and a quantitative clonogenicity test may beconducted for each of them. Thus, strictly clonal secondary culturesand/or low density cultures allowing the growth of individualizedcolonies may for example be produced.

Essentially, within the scope of the present invention, the initialbiological sample is a sample of healthy or diseased epithelial tissue,for example obtained by biopsy in a mammal, preferably in humans. Inparticular, the tissue sample may be selected from epithelia, forexample the interfollicular epidermis of adult or neonatal human skin,the cornea, mucosas, hair follicles. Samples of diseased epithelialtissues are for example obtained by biopsy of patients affected with agenetic disease (such as xeroderma pigmentosum, bullous epidermolyses,etc.), by biopsy of cicatricial skin (notably in badly burnt persons).The diseased epithelial tissues may also be tumoral tissues (carcinomas,etc.).

The biological sample may possibly comprise cells from epithelial(notably keratinopoietic) differentiation of pluripotent stem cellsselected from embryonic, fetal and induced pluripotent stem cells. Asexamples, mention will be made of cells having epithelial potential,stemming from fetal stem cells: cells from the ectodermal embryoniclayer, cells from epithelial tissues, keratinocytes, etc. The cellshaving epithelial potential stemming from so-called “induced”pluripotent stem (IPS) cells are generated by reprogramming cellsstemming from adult tissue which may be differentiated cells.

Advantageously, the epithelial cells directly extracted from thebiological sample are single healthy or diseased cells selected fromprogenitor cells, stem cells, keratinocytes.

Another object of the present invention relates to a clonal culturesystem (or to a kit comprising such a system) obtained by applying atleast steps a) to c) of the method according to the invention. Thesesystems have particular properties inherent to the fact that they derivefrom clonal cultures. The kits may for example be diagnostic kits, testsfor evaluating biological activity, toxicity tests, etc. It is quiteclear for one skilled in the art that the terms of “test”, “kit” andpossibly “system” may be equivalent here depending on the context inwhich they are used.

In a particular embodiment, a clonal culture system for epithelial cellsoptimized for evaluating and exploiting specific properties of a singlecell, comprises, within the context of the invention, a culture supportin which at least one clonal culture is sown with a single epithelialcell directly extracted from a biological sample of epithelial tissueaccording to the steps a) to c) of the method described earlier.

The present invention also relates to applications of the method and touses of the various means (system, kit, cell bank, tissue, biochip)described above.

Preferred examples of such uses and applications are:

for identifying and selecting agents having a biological activity ofinterest. An “agent” may be a candidate molecule which is tested for itsbiological activity and which is selected depending on the applications,said activity may be positive (for example, for selecting effectors ofpharmaceutical, therapeutic, cosmetic interest, etc.) or negative (forexample, for selecting toxic molecules). Alternatively, an “agent” maybe of a non-chemical nature, for example UV rays, visible light,ionizing radiations, magnetic waves, etc.;

for treatment and/or diagnosis (notably in vitro) and/or prognosis(notably in vitro) in the field of cell and/or gene therapy, notably inthe field of grafts;

for studying the behavior and/or the structural and/or functionalindividual properties specific to a single cell, a cell sub-type, a cellsub-population, or a cell population;

for producing one or more tools of functional genomics, which arenotably useful for inducing phenomena of gain or loss in biologicalactivity, for medical purposes and/or in any type of functionalexploration. For example, mention will be made of interfering RNAs,over-expression and/or repression, viral or non-viral vectors, etc.;

for evaluating the efficiency of agents having biological activity suchas molecules of pharmaceutical or cosmetic interest, and/or evaluatingthe efficiency of treatments with such agents (molecules or other typesof stimuli, for example waves, light, radiations, physical parameters,etc.).

Eventually, the various objects of the present invention as comparedwith the presently available means and methods have the followingconsiderable advantages:

(i) The possibility of producing parallel microculture series, initiatedfrom a single cell, which in practice allows the application of largescale test campaigns targeting rare sub-populations, poorly representedwithin tissues, which cannot be contemplated in standard models whichare great consumers of cell material.

(ii) The possibility of initiating clonal cultures from individuallysown cells of a selected phenotype, for example in microwells,immediately after being selected from the tissue, which gives thepossibility of contemplating the setting into place of test strategieson cell material which has not undergone a multiplication step inculture, and therefore is less likely to have been modified byartificial culture parameters before conducting the tests, in particularwhen the treatment or stimulus is immediately applied after sowingmicrocultures.

(iii) The fact of having access for the first time to the behavior ofcells individually placed in culture, which provides the possibility ofdescribing and quantifying the biological properties and the specificresponses of the different cells forming a population of interest,allowing analysis of cell heterogeneity, which is not possible instandard models which use cell populations globally.

Thus, the various objects of the present invention prove to be useful invery many fields. In addition to the examples of applications alreadydescribed above, mention may notably be made in a non-limiting way of:

(i) Evaluation of the efficiency of agents having a biological activity:function gains.

-   -   Evaluation of the efficiency of compounds bearing a beneficial        biological activity (for example, molecules of pharmaceutical        interest, cosmetic actives):

Conducting screening campaigns allowing quantification of the effects ofbiological actives at the scale of a single isolated cell: action of atreatment on the actual target cell, impact on its offspring.

Possibility of developing strategies of evaluation tests targeted onpoorly represented populations, such as normal or pathological stemcells.

-   -   Examples:

test of effectors capable 1) of inducing multiplication of stem orprogenitor cells of pluristratified epithelial tissues, 2) promotingmaintenance of the stem nature in culture in the offspring of isolatedstem cells;

test of novel anti-cancer molecules on stem cells isolated fromcarcinomas.

(ii) Problems of toxicology: function losses, cancer transformation.

-   -   Estimation of the impact of stress and toxic agents at the scale        of treated cells in isolation, after selection on the basis of        specific criteria. These tests may selectively be applied to the        cells responsible for long term renewal (stem cells) and short        term renewal (progenitors) of epithelial tissues.    -   They also allow depending on the type of targeted cells, the        design of tests adapted to prognosing and distinguishing acute        or belated deleterious effects on a tissue or organ:

Conducting toxicological tests allowing estimation of the innocuousnessof a treatment or, on the other hand, quantification of its toxiceffects: short term effect on the actual isolated cell, consequences onits offspring.

Evaluating the impact of genotoxic aggressions: at the scale of cellsstudied in isolation, acquisitions of damages to DNA, transmission ofmutations and/or genetic abnormalities to the offspring, consequences onorganogenesis, etc.

(iii) Technology of biochips: parallel/massively parallel models ofquantification and qualification of biological responses.

Screens of functional genomics on two-dimensional cell cultures and/orthree-dimensional organotypic models.

High throughput screening of molecules bearing a biologicalactivity/detection of deleterious properties (“high throughputscreening” [HTS]).

(iv) Cell and gene therapy.

-   -   Qualification of samples of cells intended to be grafted, and/or        intended to be used for producing grafts of rebuilt tissues:

Tests in culture allowing an estimation of the regenerative potential ofcells intended for clinical use: growth potential individually estimatedon cells under a clonal condition.

Prognosis tests of the capacity of engraftment of tissues rebuilt invitro: estimation of maintenance or loss of growth potential of cellsused for producing grafts.

-   -   Validation of gene transfer protocols in a clinical perspective:

Evaluation of the efficiency of a genetic correction protocol:estimation of the frequency of cells actually corrected at the end ofthe gene transfer method.

Evaluation of the stability of genetic correction: transmission to theoffspring of individualized cells.

(v) Cell and tissue engineering.

Cell systems and organotypic models compatible with the application oftests on cells followed in isolation and their offspring:

Parallel production of cell banks, each formed by the offspring of asingle cell placed in culture and in isolation.

Models of normal or pathological tissues rebuilt in vitro generated fromthe offspring of a single cell placed in culture under a clonalcondition.

The following figures illustrate, in connection with examples below,embodiments of the present invention:

FIG. 1: a diagram illustrating an embodiment of the method according tothe invention;

FIG. 2: a graphic illustration of the result of a long term expansionexperiment for producing banks of multiple keratinocytes, each stemmingfrom the offspring of a single cell;

FIG. 3: results of an experiment for producing multiple rebuiltepidermises, each stemming from the offspring of a single cell;

FIG. 4: results of the evaluation of short term clonal growth of basalkeratinocytes Itg∝6^(strong) placed in culture individually;

FIG. 5: results of an experiment in which long term cultures initiatedfrom basal keratinocytes Itg∝6^(strong) individually placed in cultureare quantified;

FIG. 6: a graphic illustration of the results of an experiment where theimpact of irradiation on epidermal keratinocytes of distinct phenotypeswas quantified at the scale of a single isolated cell;

FIG. 7: results of an experiment in which the functional test ofparallel clonal microcultures was used for evaluating the consequencesof irradiation carried out on an isolated cell, on the growth potentialof its offspring, and in which the behavior of epidermal keratinocytesof distinct phenotypes was compared;

FIGS. 8A, 8B, 8C: result of a search for abnormalities at the chromosome10 by CGH chips:

-   -   Offspring of two non-irradiated keratinocytes: K1 and K2    -   Offspring of an irradiated keratinocyte: K3.    -   Particular embodiments and advantages of the present invention        are described in the exemplary embodiments below, which deal        with keratinocytes from adult human interfollicular epidermis.

These examples are provided purely as an illustration; they do not limitby any means the object and scope of the invention.

EXAMPLES A—Experimental Procedures

A-1—Preparation of the Cell Material

A-1-1—Epithelial Cells Intended to be Used for Clonal Cultures

The tissue biopsies which in the example described here are biopsies ofadult human skin, were first of all decontaminated, for example bysoaking them in a physiological solution containing betadine. In orderto allow separation between the epithelial tissue and the associatedconnective tissue (in the present case, the epidermis and dermis), thesamples were then incubated in an enzyme solution at 4° C. for 10-15hours (Gibco trypsin). At the end of this enzymatic digestion step, thetissue samples were dissected with fine tweezers, so as to isolate theepithelial portion of the tissue (here, the interfollicular epidermis).The enzymatic treatment completed by a mechanical dissociation step bysuctions and discharge with a pipette, allows extraction of thekeratinocytes which make up the fragments of epithelia. The cellsuspension was finally filtered on a sieve with a mesh of 50-70 microns(BD Falcon), in order to remove the cell aggregates. At the end of thesesteps, the cell samples appear as monocellular suspensions, which may beused for sowing clonal cultures.

A-1-2—Fibroblasts Used as Supporting Cells

In the described examples, epithelial cells (keratinocytes obtained frominterfollicular epidermis) were studied under a clonal condition in aculture system using as support a nutritive layer of fibroblasts madeunable to multiply by gamma irradiation with a dose of 60 Grays. Thus,these cells remained static but live, and they supported the growth ofthe studied epithelial cells. These fibroblasts may notably be extractedfrom the dermal portion of skin biopsies. To do this, dermis fragmentswere incubated in an enzymatic solution consisting of a mixture ofdispase (Roche) and of collagenase (Roche) for 2-4 hours at 37° C.Digestion by the enzymes, combined with mechanical stirring, allowsextraction of the fibroblasts. After removing non-digested tissuefragments by filtration on a sieve (BD Falcon), and then by washing, theobtained fibroblasts were then placed in culture in a medium consistingof 90% DMEM (Gibco) and 10% serum of bovine origin (Gibco), in order tobe amplified. After multiplication in culture, the fibroblasts wereirradiated, and then frozen so as to be stored until use.

A-2—Immuno-Phenotypic Labellings

Epithelial cells used for illustrating certain embodiments of theinvention are keratinocytes having a strong expression level of α6integrin (Itgα6 or CD49f) and a weak expression level of the receptor oftransferrin (CD71): phenotype Itgα6^(strong) CD71^(weak). For achievingthe labeling with fluorescent antibodies required for defining thisphenotype, the cell samples were placed in suspension in physiologicalsaline buffer (PBS) supplemented with 2% bovine albumin serum (SAB)(Sigma), and then incubated for 10 minutes at 4° C. with mouseimmunoglobulins (Jackson Immuno-Reasearch), in order to saturate thenon-specific binding sites of the antibodies. Labelling of the antigensCD49f and CD71 was then carried out by adding specific antibodiescoupled with fluorochromes, and then by incubation for 30 minutes at 4°C.: anti-CD49f-PE (phycoerythrin) (clone GoH3, BD Pharmingen) andanti-CD71-APC (allophycocyanin) (clone M-A712, BD Pharmingen). Afterwashing the antibodies in excess, the samples were ready to be used forsowing clonal cultures.

A-3—Automated Sowing of Clonal Culture Microcultures

A-3-1—Sowing

In the described exemplary embodiments, the clonal microcultures ofkeratinocytes were sown in an automated way with a flow cytometerequipped with a cloning module (MoFlo, Cytomation). Excitation of thefluorochromes coupled with the labelling antibodies was carried out byusing a 488 nm argon laser (Coherent) and a 630 nm laser diode. Thesignals emitted by phycoerythrin (PE) and allophycocyanin (APC) wererespectively detected and quantified in wavelength windows of 580±30 nmand 670±30 nm. The sorting criterion selected in the present casecorresponded to keratinocytes having the phenotype Itgα6^(strong)CD71^(weak) and accounting for about 1% of the total keratinocytes: asub-population of keratinocytes described as being enriched in epidermalstem cells (Li et al., 1998).

A-3-2—Quality Control of the Clonal Sowings

In parallel with the clonal cultures intended to be used for the testsand studies (culture conditions described hereafter), series of clonalsowings were carried out with the purpose of validating the procedurefor depositing the cells. These depositions were carried out understrictly identical technical conditions, but in a medium more favourablefor locating the cells than the one used for their growth. Thisobservation medium may for example be a physiological saline buffer(PBS) added with Hoechst 33342 (Sigma) with a concentration of 10micrograms/ml. The Hoechst is a DNA coloring agent which is fluorescentunder UV-excitation. Under these conditions, the individually sown cellsin a large number of culture wells may be located, which allowsverification of the efficiency of the method. Each microculture shouldcontain a single cell, never two and the frequency of empty wells shouldbe minimized.

A-4—Culture Conditions

A-4-1—Primary Clonal Growth

In the described examples, microcultures of keratinocytes were carriedout in culture plates comprising 96 wells in which collagen of type Iwas adsorbed (Biocoat, Becton-Dickinson). The day before the sowing ofepithelial cells under a clonal condition, a nutritive layer ofirradiated fibroblasts was set into place in the culture wells. Thesesupporting cells were sown at a density of 6,000 cells/cm². The culturemedium used for growing the keratinocytes was based on a mixture of DMEM(Gibco) medium and of Ham F12 (Gibco) medium, added with serum of bovineorigin (Hyclone). This basic medium was notably supplemented with EGF(Chemicon), with insulin (Sigma), with hydrocortisone (Sigma), withadenine (Sigma), with triiodothyronine (Sigma), with L-glutamine(Gibco), and with a solution of antibiotics and antimycotics (Gibco).After automated sowing of the keratinocytes in an amount of one singlecell per well, the cultures were maintained in culture at 37° C. in anatmosphere comprising 90% humidity, in the presence of 5% CO₂, in thepresent case for 2 weeks. At the selected time, counting of the wells inwhich a cell clone had developed was carried out, and the number ofkeratinocytes forming each clone was then determined individually, afterdetaching the cells by trypsination (Gibco).

A-4-2—Study of the Tissue Reconstruction Potential

In the described examples, the tissue reconstruction potential of theoffspring of keratinocytes initially placed in culture individually wasdemonstrated in an epidermal reconstruction model on de-epidermized deadhuman dermis (Regnier et al., 1986). For preparing dermal supports,human skin samples were incubated for 10 days at 37° C. in PBS buffer,in order to detach the epidermis from them, which was then removed. Theepidermis-free dermal samples were cut into squares of about 1 cm². Theywere then subject to several successive freezing/thawing cycles whichled to the killing of the dermal cells. The obtained a cellular dermiseswere stored at −20° C. until use. The process for rebuilding athree-dimensional epidermis comprised 2 successive culture steps. Thecell samples from clonal microcultures were first of all sown on thedermal supports and cultivated for 1 week in immersion in a comparableculture medium similar to the one used for the primary clonal culture(composition example described above). The second step of the epidermalreconstruction method consisted in placing the epidermises being formedat the interface between the liquid medium and the ambient air of theincubator. Cultivation was then continued for 1-2 weeks before reachingcomplete differentiation. The histological characteristics of therebuilt three-dimensional tissue were viewed after fixing and stainingwith hemalum-erosine-safran (HES).

A-4-3—Evaluation of the Long Term Expansion Capacity

The keratinocytes from clonal microcultures were detached bytrypsination (Gibco), and then placed in a mass culture, individuallyfor each studied clone. These cultures were carried out on plasticsurfaces on which collagen of type I was adsorbed (for example Petri,Biocoat, Becton-Dickinson plates). The culture conditions wereequivalent to those used for primary clonal growth: a nutritive layer ofirradiated fibroblasts, a culture medium with similar composition. Afterone week, the cultures reached 50%-80% confluence. The keratinocyteswere then detached by trypsination, counted and then resown at a densityfrom 2,000 to 3,000 cells/cm², under identical conditions. The cultureswere then transplanted and sub-cultivated every week, until exhaustionof the expansion potential of the cells. At the end of the growth stepof the primary clone and of the successive sub-cultures, cellproliferation was estimated in terms of an expansion coefficient and ofthe number of achieved population doublings, in order to quantify thetotal expansion potential of keratinocytes initially placed in cultureindividually. Number of population doublings=(Log N/N₀)/Log 2

N₀=Number of sown cells

N=Number of cells obtained at the end of the culture step.

A-4-4—Analysis of the Secondary Clone-Forming Capacity

The question is of estimating maintenance or loss of the potential togenerate colonies from cells forming the offspring of clonedkeratinocytes. To do this, the cells of primary clones were detached bytrypsination (Gibco), and then sown at low density so as to obtaingrowth of colonies separate from each other (for example, 5 cells/cm²)under conditions similar to those described above for evaluating thelong term expansion potential. After 14 days, the cultures were fixedwith 70% ethanol, dried and then stained by two successive baths ineosin (RAL reagents) and in Blue RAL 555 (RAL reagents). The parameterstaken into account for quantifying the clone-forming capacity of thestudied cells notably were the number and size of the obtained colonies.

B—Performance of the Means of the Invention

A parameter used for analyzing the growth potential of epithelial cellsis their capacity of generating colonies in a low density culture (CFEfor “colony-forming efficiency”) or clones when these are cultures sownwith a single cell (CFE for “clone-forming efficiency”). It is quiteobvious that the more the methods allow demonstration of high CFEvalues, the more they will be useful for effectively quantifying thegrowth potential of epithelial cells.

Table I below shows the results obtained according to two known methodsfor selecting and cultivating keratinocytes (they are conventionallyused in the laboratory and have been described in publications), as wellas the results obtained according to the method of the invention. TABLEI Phnotype Type of of the cell Obtained Study cells used Clonalitymaterial CFE values* Larderet et ‘Side No Cells from 14.5% al., (2006)population’ a culture (SP) Rachidi et strong α6 No Cells 9.9% al. (2007)integrin/ extracted weak CD71 from tissue Method strong α6 Yes Cells48.4%** according integrin*** extracted to the from tissue presentinvention*CFE = % of cells capable of giving rise to a colony (low densityculture situation) or to a clone (cultures with only 1 cell per well).**Average calculated over 3 conducted experiments from independentsamples.***In a non-exclusive way.

C—Exemplary Embodiment No. 1

The use of the model of parallel clonal microcultures for producingbanks of multiple keratinocytes, each from the offspring of a singlecell (FIG. 2).

C-1—Materials and Methods

Extraction of keratinocytes from an epidermis from an adult skin biopsy(mammary reduction), dissociation and suspension.

Labelling of the suspended keratinocytes with antibodies coupled withfluorochromes giving the possibility of sorting a phenotype of “stem”cells (strong expression level of integrin α6 (Itgα6) and low expressionlevel of the receptor of transferrin (CD71): phenotype Itgα6^(strong)CD71^(weak); selection model: Li et al., 1998).

With a cloning module by flow cytometry, automated sowing of multi-wellculture plates, in an amount of one single “stem” phenotype cell perwell.

After 2 weeks of culture, localization of the wells in which the clonedkeratinocyte has given rise to a cell clone.

Sowing mass cultures from each clonal microculture, under conditionspromoting cell multiplication.

Successive transplantations of the cultures in order to obtain theoffspring of the initial cloned cells at different amplification stages(for example: a “young” or “old” culture state), and an amount of cellmaterial stemming from the cloned cells compatible with the forming ofbanks of frozen cells.

Test of the maximum number of successive sub-cultures which it waspossible to achieve for each culture initiated from a singlekeratinocyte and estimation of the total accumulated number ofkeratinocytes produced at the end of the long term cultures.

C-2—Results

In a cohort of 5 cell clones tested for their capability of generating abank of keratinocytes, all of them were able to be sub-cultivated andtheir offspring sufficiently amplified in order to be frozen.

Two weeks after initiation of the cultures, the 5 selected clonesconsisted of 8.64×10⁴ to 1.11×10⁵ keratinocytes, which is equivalent to16.40 to 16.86 successive cell generations achieved since the stage ofthe single cloned cell.

1 clone generated an accumulated offspring of 8.48×10¹² keratinocyteswithin 8 weeks of multiplication in culture (No. 1), which is equivalentto an accumulation of 42.95 average population doublings.

2 clones generated an accumulated offspring of 6.36×10¹¹ and 2.4×10¹¹keratinocytes (No. 2 and No. 3), which is equivalent to 39.21 and 37.80average population doublings respectively.

1 clone generated an accumulated offspring of 2.03×10¹⁰ keratinocyteswithin 6 weeks of multiplication in culture (No. 4), which is equivalentto an accumulation of 34.24 average population doublings.

1 clone generated an accumulated offspring of 2.15×10⁹ keratinocyteswithin 6 weeks of multiplication in culture (No. 5), which is equivalentto an accumulation of 31.00 average population doublings.

C-3—Conclusion

The model of parallel clonal microcultures according to the presentinvention represents a technology allowing standardized generation ofmultiple banks of keratinocytes, each from the offspring of a singlecell directly isolated from a tissue sample.

It also allows estimation and comparison, at the scale of the individualcell, of the proliferation potential of distinct cell types (forexample: progenitors, epidermal stem cells).

D—Exemplary Embodiment No. 2

The use of the system of clonal microcultures for producing multiplerebuilt epidermises, each from the offspring of a single cell (FIG. 3).

D-1—Materials and Methods

Extraction of keratinocytes from an epidermis from adult skin biopsy(mammary reduction), dissociation and suspension.

Labelling the suspended keratinocytes with antibodies coupled withfluorochromes giving the possibility of sorting in flow cytometry aphenotype of “stem” cells (strong expression level of integrin α6(Itgα6) and low expression level of the receptor of transferrin (CD71);phenotype Itgα6^(strong) CD71^(weak); selection model: Li et al., 1998).

By means of a cloning module by flow cytometry, automated sowing ofmulti-well culture plates, in an amount of one single “stem” phenotypecell per well.

After 2 weeks of culture, localization of the wells in which the clonedkeratinocyte has given rise to a cell clone.

The use of keratinocytes forming the cell clones separately for each ofthem, in order to produce rebuilt epidermises (for example:reconstruction of an epidermis on de-epidermized dead human dermis:Regnier et al., 1986).

D-2—Results

A cohort of 5 cell clones was tested for the individual capability ofeach clone of generating a three-dimensional rebuilt epidermis.

The experiment was conducted at a growth stage of the clones equivalentto the one described in the exemplary embodiment No. 1 (multiplicationcorresponding to ˜16 to 17 successive cell generations).

The 5 tested clones prove to be capable of producing an epidermis havingan organization representative of that of a native epidermis.

D-3—Conclusion

The technology of parallel clonal microcultures according to theinvention allows production of series of rebuilt epidermises, theparticularity of which is of each being from the offspring of a singlecell, while the conventionally used models for large scale testcampaigns are generated from banks from a mixture of cells.

The rebuilt epidermises produced according to the method object of thepresent invention are generated from a cloned cell immediately afterextraction from the tissue, and not after a multiplication step inculture, likely to modify the characteristics thereof.

In this exemplary embodiment, the clones were used for epidermalreconstruction at a growth stage corresponding to ˜16-17 successive cellgenerations. In other embodiments, epidermal reconstructions may beachieved from an earlier or more belated growth stage of the clonedcells.

The culture model according to the present invention therefore providesan original functional test allowing qualification of the organogenesispotential of cells initially placed in culture individually, immediatelyfollowing selection from the tissue. It provides the possibility ofevaluating the impact of a stimulus or stress at various growth stagesof cells placed in culture in isolation, and then of studying theconsequences thereof on the capacity of tissue reconstruction in theshort or medium term after treatment.

E—Exemplary Embodiment No. 3

The use of the model of parallel clonal microcultures for characterizingthe clone-forming capacity of keratinocytes present at a tissue locationof interest. In particular, the question is of estimating theircapability of giving rise to a cell clone, the size of which representsan indicator of their short term multiplication potential (FIG. 4).

E-1—Materials and Methods

-   -   Extraction of keratinocytes from an epidermis from an adult skin        biopsy (mammary reduction), dissociation and suspension.    -   Labelling the suspended keratinocytes with an antibody coupled        with a fluorochrome giving the possibility of sorting a        population of keratinocytes corresponding to the basal layer of        the epidermis (strong expression level of integrin α6 (Itgα6):        phenotype Itgα6^(strong)).    -   By means of the cloning module by flow cytometry, automated        sowing of multi-well culture plates, in an amount of one single        basal keratinocyte per well.    -   After 2 weeks of culture, localization of the wells in which the        cloned keratinocyte has given rise to a cell clone, and then        detachment and counting of the keratinocytes, individually for        each clone.    -   Classification of the different clones according to their        individual size.        E-2—Results

The analysis of the distribution of the sizes of clones generated bykeratinocytes from the basal layer of the epidermis, conducted on anaccumulated cohort of ˜800 clones, reveals the functional heterogeneityof this cell compartment (FIG. 4).

At the top of this hierarchy, a minority fraction of clones is found,characterized by a significant capability of short term proliferation,the size of which may exceed 15×10⁴ keratinocytes within 2 weeks.

-   -   At the bottom of the hierarchy, on the contrary, a fraction of        clones is found characterized by a very limited capability of        proliferation. The size of these abortive clones does not exceed        10⁴ keratinocytes after 2 weeks of culture.    -   Between these extremes, the majority of the clones are found,        which appear to be distributed according to a size gradient, the        size value represented in majority being located around 9×10⁴        keratinocytes.        E-3—Conclusion    -   The technology of parallel clonal microcultures according to the        invention allows specific estimation of the individual        clone-forming capability of cells from a sample of interest, in        this exemplary embodiment, a preparation of basal keratinocytes        of the epidermis.    -   In particular, the method proves to be resolvent in order to        define a clonal growth profile providing a ‘functional        signature’ representative of a tissue, or a sub-localization        profile within a tissue, in the present case, the basal layer of        the adult human interfollicular epidermis.    -   Further, with the system, it is possible to distinguish, within        a cell sample of interest, cells having distinct potentialities        depending on their short term clonal growth capacity.

The culture model according to the present invention therefore providesan original functional test allowing estimation of the clone-formingcapacity of cohorts of cells individually placed in culture. A possibleapplication is the development of quality controls achieved at the scaleof the individual cell aiming at evaluating the functionality (ornon-functionality) of cell samples of interest, by comparison with avalidated reference. The system of clonal microcultures further providesthe possibility of generating cell samples from a single cell, eachindividually corresponding to a specifically defined short termproliferation capacity. Another possible application consists of usingthe system for conducting studies aiming at analyzing the short termfunctional consequences of a (beneficial or toxic) treatment applied atthe scale of the individual cell.

F—Exemplary Embodiment No. 4

The use of the model of parallel clonal microcultures for characterizingthe long term growth potential of keratinocytes from a sample ofinterest. In particular the question is of detecting the presence ofkeratinocytes having one of the functional properties associated withepidermal stem cells, i.e. the capability of carrying out at least 100population doublings in culture (FIG. 5).

F-1—Materials and Methods

-   -   Extraction of keratinocytes from an epidermis from an adult skin        biopsy (mammary reduction), dissociation and suspension.    -   Labelling of the suspended keratinocytes with an antibody        coupled with a fluorochrome giving the possibility of sorting a        population of keratinocytes corresponding to the basal layer of        the epidermis (strong expression level of integrin α6 (Itgα6):        phenotype Itgα6^(strong)).    -   By means of the cloning module by flow cytometry, automated        sowing of multi-well culture plates, in an amount of one single        basal keratinocyte per well.    -   After 2 weeks of culture, localization of the wells in which the        cloned keratinocyte has given rise to a cell clone.    -   Sowing of mass cultures from a representative cohort of clonal        microcultures, under conditions promoting cell multiplication.    -   Successive transplantations of the different cultures        separately, every week, until the limit of their individual        multiplication potential is reached.    -   Evaluating the number of successive sub-cultures which each        culture of clonal origin was capable of sustaining and        estimating the total accumulated number of population doublings        carried out at each transplantation and at the end of the long        term cultures.        F-2—Results

The analysis of the long term growth potential of a cohort of 23 clonesfrom basal keratinocytes reveals marked potential heterogeneity of thiscompartment (FIG. 5).

-   -   At the bottom of the observed potential hierarchy are found        clones having a restricted growth potential, which can only be        maintained in culture for 6-7 weeks and only capable of carrying        out a total of 30-40 population doublings.    -   At the top of the potential hierarchy are found clones having a        very large growth potential which may be sub-cultivated for more        than 24 weeks without notable reduction of their growth        capability and thus capable of exceeding the expansion level of        100 population doublings.    -   Between these extremes are found clones distributed according to        a wide potential gradient, which may be sub-cultivated for 9-18        weeks and in majority capable of carrying out 40-80 population        doublings.        F-3—CONCLUSION    -   The technology or parallel clonal microcultures according to the        invention allows specific estimation of the long term growth        potential of the cells from a sample of interest, in this        exemplary embodiment, a preparation of basal keratinocytes of        the epidermis.    -   In particular, the method proves to be resolvent in order to        distinguish clones generated from a stem cell a posteriori by        their capability of carrying out accumulation of at least 100        population doublings, from clones stemming from a progenitor        cell, the long term proliferation capacity of which is more        limited, generally comprised between 30 and 80 population        doublings.    -   With the system, it is possible to generate cell samples        corresponding to the offspring of stem cells and of progenitor        cells, the properties of which may be studied and compared to        different phases of their long term proliferation.

The culture model according to the invention therefore provides anoriginal functional test allowing qualification of the long term growthpotential of cells initially placed in culture individually. For exampleit provides the possibility of estimating the regenerative potential ofa sample, notably by evaluating the presence (or the absence) of stemcells. A possible use consists of conducting studies aiming at analyzingthe long term functional consequences of a (beneficial or toxic)treatment applied at the scale of the individual cell.

G—Exemplary Embodiment No. 5

The use of the functional test of parallel clonal microcultures forquantifying at the scale of the single isolated cell the impact ofirradiation on epidermal keratinocytes of distinct phenotypes (FIG. 6).

G-1—Materials and Methods

-   -   Extraction of keratinocytes from an epidermis from an adult skin        biopsy (mammary reduction), dissociation and suspension.    -   Labelling of the suspended keratinocytes with antibodies coupled        with fluorochromes giving the possibility of sorting in flow        cytometry a phenotype of “stem” cells (strong expression level        of integrin α6 (strong expression level of integrin α6 (Itgα6)        and weak expression level of the receptor of transferrin (CD71):        phenotype (Itgα6^(strong) and CD71^(weak)) and a phenotype of        “progenitor cells” (strong expression level of integrin α6        (Itgα6) and strong expression level of the receptor of        transferrin (CD71): phenotype Itgα6^(strong) CD71^(strong))        (selection model: Li et al., 1998).

By means of a cloning module by flow cytometry, automated sowing ofmulti-well culture plates, in an amount of one single cell per well, andthis for each of the two studied cell phenotypes.

Twenty hours after sowing, irradiation of each isolated cell with asingle dose of 2 Gy (γ radiation).

After 2 weeks of culture, counting the wells in which a cell clone hasdeveloped, and then detachment and countings of keratinocytes,individually for each clone.

G-2—Results

G-2-1—Impact of Irradiation on the Clone-Forming Capacity of IsolatedKeratinocytes (FIG. 6A)

Under a control condition (without irradiation), both tested phenotypesof keratinocytes exhibited strong capability of generating a cell clone.They were not notably distinguished on this criterion.

-   -   70.3% of individually sown keratinocytes of phenotype        Itgα6^(strong) CD71^(weak) (sub-population enriched in stem        cells) gave rise to a cell clone.    -   61.7% of keratinocytes of phenotype Itgα6^(strong) CD71^(strong)        (sub-population composed of progenitors) generated a clone.

The keratinocytes of both phenotypes were on the other hand differentlyaffected by irradiation.

-   -   The percentage of keratinocytes of phenotype Itgα6^(strong)        CD71^(weak) giving rise to a clone was lowered from 70.3%        (control condition) to 47.3% (irradiation of 2 Gy), which        allowed maintenance of the clone-forming capacity of these cells        to be estimated at 67.3%.    -   The clone-forming capacity of keratinocytes of phenotype        Itgα6^(strong) CD71^(strong) was drastically reduced by        irradiation: 61.7% of cells giving rise to a clone under a        control condition versus 23.0% under an irradiation condition,        which allowed maintenance of the clone-forming capacity of these        cells to be estimated at only 37.3%.        G-2-2—Impact of Irradiation on the Growth Potential of the        Clones (FIG. 6B)

Under a control condition, the size of the produced clones has notproved either to be a criterion allowing clear distinction of both typesof studied keratinocytes.

The keratinocytes of both phenotypes have proved to be capable ofgenerating a large proportion of clones of large size comprising atleast 5×10⁴ keratinocytes.

The analysis of the distributions of the size of the obtained clonesprovided parameters allowing clear distinction of the specific responsesto irradiation of keratinocytes of distinct phenotypes.

Moderate reduction of the capacity of cloned keratinocytesItgα6^(strong) CD71^(weak) of generating large size clones followingirradiation (median size of the clones under a control condition:10.1×10⁴ cells/clone; median size for the irradiated group: 6.8×10⁴cells/clone).

Strongly marked reduction of the capacity of keratinocytesItgα6^(strong) CD71^(strong) of producing large size clones followingirradiation (median size of the clones, control group: 8.5×10⁴cells/clone; median size for the irradiated group: 1.2×10⁴ cells/clone).

G-3—Conclusion

The model of the parallel clonal microcultures according to theinvention proves to be performing for analyzing the growth capacity ofkeratinocytes of specific phenotypes at the scale of the individualcell. Indeed, as the values of clone-forming efficiencies obtained inthis model reach 60-70% of the cloned cells, they prove to be verysuperior to what is generally described in conventional culture systems,concerning keratinocytes directly stemming from tissue biopsy, for whichthe values are of the order of 10%.

This model also proves to be performing for detecting, qualifying andquantifying a deleterious effect on the cell growth potential. Thepresent example illustrates the capability of the system of being valuedby the development of radiotoxicology tests in vitro.

H—Exemplary Embodiment No. 6

The use of the functional test of parallel clonal microcultures forevaluating the consequences of irradiation of a single cell on thegrowth potential of its offspring. Comparison of the behavior ofepidermal keratinocytes with distinct phenotypes (FIG. 7).

H-1—Materials and Methods

Following the procedure described in the scope of the exemplaryembodiment No. 5:

Low density sowing of a portion of the keratinocytes stemming from theclones, in order to obtain individualized colonies: in this example,density of 5 keratinocytes/cm².

After 2 weeks of culture, fixation and staining of the colonies, so asto be able to carry out a microscopic and macroscopic observation.

H-2—Results

Under a control condition (without irradiation of the initial cellsplaced in culture individually), the groups of clones tested for theircapacity of generating secondary colonies have shown very similarcharacteristics for this criterion.

The capacity of producing secondary colonies from the 2 series ofclones, respectively stemming from keratinocytes Itgα6^(strong)CD71^(weak) [clones (1) to (5)] and from keratinocytes Itgα6^(strong)CD71^(strong) [clones (11) to (15)] has proved to be comparable.

Both of these groups comprised both cell clones abundantly giving riseto secondary colonies of large size [for example: clones (1) and (11)]and clones giving rise to not very abundant colonies and of small size[for example: clones (5) and (15)].

As regards groups of clones stemming from a cell having undergoneirradiation (single dose of 2 Gy), the capacity of generating secondarycolonies proves to be clearly distinct for the 2 phenotypes of thetested keratinocytes.

The group of clones stemming from keratinocytes Itgα6^(strong)CD71^(weak) [clones (6) to (10)] exhibited a capacity of producingsecondary colonies equivalent to that of the non-irradiated groups.

On the other hand, the group of clones stemming from keratinoytesItgα6^(strong) CD71^(strong) having been subject to irradiation [clones(16) to (20)] exhibited reduced secondary clone-forming capacity (lossesof colonies with a diameter ≧5 mm).

H-3—Conclusion

The model of the parallel clonal microcultures according to the presentinvention is adapted for demonstrating, qualifying and quantifying thenon-immediate consequences of irradiation carried out on individuallystudied keratinocytes. In the present case, the demonstrated deleteriouseffect was a loss of growth capacity measured on the offspring of cellsplaced in a clonal culture.

I—Exemplary Embodiment No. 7

The use of the model of parallel clonal microcultures for analyzing thelong term consequences of genotoxic stress applied on cells placed undera clonal condition. The question is of applying stress for a few hoursafter sowing the cells in separate culture wells individually, and thenof initiating long term cultures after the cells have divided. Thepresence of abnormalities at the level of the genome is then sought atthe level of the offspring of the cloned cells, after the latter hascarried out a determined number of population doublings. This search isfor example carried out by using a technique allowing detection ofrepresentation disequilibria of DNA sequences in the genome (deletions,amplifications) comparative genomic hybridization (CGH) (FIG. 8).

I-1—Materials and Methods

-   -   Extraction of keratinocytes from an epidermis stemming from an        adult skin biopsy (mammary reduction), dissociation and        suspension.    -   Labelling of the suspended keratinocytes with an antibody        coupled with a fluorochrome giving the possibility of sorting a        population of keratinocytes corresponding to the basal layer of        the epidermis (strong expression level of the integrin α6        (Itgα6): phenotype Itgα6^(strong)).    -   By means of the cloning module by flow cytometry, automated        sowing of multi-well culture plates, in an amount of one basal        keratinocyte per well.    -   Separation of the clonal microcultures into 2 groups: 1) a group        subject to a single dose of 2 Grays of gamma irradiation 19        hours after sowing; 2) a non-irradiated control group.    -   After 2 weeks of culture, for each of the 2 groups, localization        of the wells in which the cloned keratinocyte has given rise to        a cell clone.    -   Sowing of mass cultures from cohorts of clonal microcultures        representative of each of the 2 groups, under conditions        promoting cell multiplication.    -   Successive transplantations of the different cultures        separately, every week, until the limit of their individual        multiplication potential is reached.    -   Evaluation of the number of successive sub-cultures which each        culture of clonal origin was capable of sustaining and        estimation of the total accumulated number of population        doublings carried out at each transplantation and at the end of        the long term cultures.    -   From the control and irradiated groups, selection of cultures        showing very significant long term proliferation capacity,        notably capable of carrying out at least 150 population        doublings.    -   For each selected candidate, preparation of genomic DNA samples        corresponding to a belated long term proliferation stage, in the        present case, the cultures having carried out about 150        population doublings after clonal sowings.    -   On the generated DNA samples, stemming from the control and        irradiated groups, the search for areas of the genome having        abnormalities of the deletion type and/or amplification by        comparative genomic hybridization (CGH) versus reference DNA        (CGH chips Constitutional Chip® 4.0, PerkinElmer, Inc.;        according to a method recommended by the manufacturer).        I-2—Results

Cytogenetic analysis by CGH chips of long term cultures of clonal originshows that the investigated gamma ray dose of 2 Grays has theconsequence that acquired chromosomal abnormalities are transmitted tothe offspring, which prove to be detectable in a large number of celldivisions after applying the genotoxic stress (FIG. 8).

-   -   In the presented example, amplification of a locus with a size        of 44.3 megabases located on the chromosome 10 (region        10q11.21-10q23.1) is detected at the level of the DNA of the        offspring of a keratinocyte, after 150 population doublings        following irradiation of the latter.    -   On the other hand, this same studied genomic segment at the DNA        level of the offspring of 2 exemplary keratinocytes which have        not been irradiated, does not exhibit this type of alteration of        the genome.        I-3—Conclusion    -   With the technology of clonal microcultures, it is possible to        subject basal keratinocytes of the epidermis individually to a        genotoxic stress, in the present case gamma irradiation, and        then to evaluate the long term consequences thereof on the        offspring.    -   In particular, the method proves to be valid for analyzing at a        clonal scale, the impact of a stress on the integrity of the        genome of the basal keratinocytes.    -   In this exemplary embodiment, the system allows detection of a        genotoxic effect of gamma irradiation on keratinocytes stemming        from the basal layer of the epidermis.

The culture model according to the present invention therefore providesan original system allowing toxicology tests to be conducted at thescale of the individual cell. For example, it provides the possibilityof characterizing the individual sensitivity of basal keratinocytes ofthe epidermis to genotoxic agents. A possible use is the conducting oftests aiming at detecting the occurrence of abnormalities at the genomeof the deletions and/or amplifications type, consecutively to exposureto a toxic agent, and analyzing their transmission to offspring duringsuccessive cell divisions, in particular in the long term.

REFERENCES

-   Gangatirkar et al., Nat. Protoc. 2007. 2: 178-186-   Li et al., Proc. Natl. Acad. Sci. USA. 1998. 95: 3902-3907-   Régnier et al., Exp. Cell Res. 1986. 165: 63-72-   Lorenz K et al. Cells Tissues Organs. 2008 Aug. 11.-   Barrandon and Green, Proc. Natl. Acad. Sci. USA. 1987. 84: 2302-2306-   Larderet et al., Stem Cells. 2006. 24: 965-974-   Rachidi et al., Radiother. Oncol. 2007. 83(3):267-76.

1. A system for clonal culture of epithelial cells optimized forevaluating and exploiting specific properties of a single cell, whereina culture support comprises at least one clonal culture sown with asingle epithelial cell directly extracted from a biological sample ofhealthy or diseased epithelial tissue.
 2. The system according to claim1, characterized in that said culture support comprises at least twoparallel clonal cultures, each of said cultures being sown with adistinct and single epithelial cell directly extracted from saidbiological sample.
 3. The system according to claim 1 or 2,characterized in that it is a biochip.
 4. A method for clonal culture ofepithelial cells optimized for evaluating and exploiting specificproperties of a single cell, comprising at least the steps of: a)extracting one or more epithelial cells directly from a biologicalsample of healthy or diseased epithelial tissue; b) optionally,selecting at least one population and/or sub-population of epithelialcells from the cells extracted in step a); c) producing a clonal culturesown with a distinct and single epithelial cell directly stemming fromstep a) or b); and d) qualitatively and/or quantitatively evaluatingcell growth in the clonal culture of step c).
 5. The method according toclaim 4, characterized in that it further comprises step e) consistingof amplifying the cell population of the clonal culture of step c), orits offspring, by one or more successive sub-cultures.
 6. The methodaccording to claim 4 or 5, characterized in that it further comprisesstep f) consisting of using the cell population of the clonal culture ofstep c), or its offspring, in order to rebuild a three-dimensionaltissue, so as to evaluate its tissue reconstruction potential.
 7. Themethod according to any of the claims 4 to 6, characterized in that itfurther comprises step g) consisting of sub-cultivating the cellpopulation of the clonal culture of step c), under conditions promotingcell expansion until exhaustion of the expansion potential, so as toevaluate the long term expansion potential of said cell population. 8.The method according to any of the claims 4 to 7, characterized in thatit further comprises step h) consisting of evaluating the clone-formingpotential of the offspring of the cell population of the clonal cultureof step c), by a quantitative clonogenicity test wherein strictly clonalsecondary cultures and/or low density cultures allowing growth ofindividualized colonies are produced.
 9. The method according to any ofthe claims 4 to 8, characterized in that step c) comprises theproduction of at least two parallel clonal cultures, each of saidcultures being sown with a distinct and single epithelial cell directlystemming from step a) or b).
 10. The system according to any of claims 1to 3, or the method according to any of claims 4 to 9, characterized inthat said biological sample of epithelial tissue is obtained by biopsyin a mammal, preferably in humans.
 11. The system according to any ofclaims 1 to 3 and 10, or the method according to any of claims 4 to 10,characterized in that said epithelial tissue is selected from epithelia,for example the interfollicular epidermis of adult or neonatal humanskin, the cornea, the mucosas, the hair follicles.
 12. The systemaccording to any of claims 1 to 3, 10 and 11, or the method according toany of claims 4 to 11, characterized in that the epithelial cell(s)directly extracted from said biological sample is(are) healthy ordiseased single cell(s) selected from progenitor cells, stem cells,keratinocytes.
 13. The system according to any of claims 1 to 3,characterized in that it is obtained by applying at least steps a) to c)of the method according to claim
 4. 14. A clonal cell bank obtainable atthe end of step e) of the method according to claim
 5. 15. Athree-dimensional tissue rebuilt from a clonal culture obtainable at theend of step f) of the method according to claim
 6. 16. The tissueaccording to claim 15, characterized in that it is selected fromepithelia, the skin, the epidermis.
 17. A biochip comprising at leastone tissue according to claim 15 or
 16. 18. The use of: the systemaccording to any of claims 1 to 3 and 10 to 13, or the cell bankaccording to claim 14, or the tissue according to claim 15 or 16, or thebiochip according to claim 17, or the application of the methodaccording to any of claims 4 to 12, for identifying and selecting agentshaving a biological activity of interest.
 19. The use of: the systemaccording to any of claims 1 to 3 and 10 to 13, or the cell bankaccording to claim 14, or the tissue according to claim 15 or 16, or thebiochip according to claim 17, or the application of the methodaccording to any of claims 4 to 12, for in vitro diagnosis and/or invitro prognosis in the field of cell and/or gene therapy, notably in thefield of grafts.
 20. The use of: the system according to any of claims 1to 3 and 10 to 13, or the cell bank according to claim 14, or the tissueaccording to claim 15 or 16, or the biochip according to claim 17, orthe application of the method according to any of claims 4 to 12 forstudying the behavior and/or the structural and/or functional individualproperties specific to a cell, a cell sub-type, a cell sub-population,or a cell population.
 21. The use of: the system according to any ofclaims 1 to 3 and 10 to 13, or the cell bank according to claim 14, orthe tissue according to claim 15 or 16, or the biochip according toclaim 17, or the application of the method according to any of claims 4to 12, for producing one or more tools of functional genomics.
 22. Theuse of: the system according to any of claims 1 to 3 and 10 to 13, orthe cell bank according to claim 14, or the tissue according to claim 15or 16, or the biochip according to claim 17, or the application of themethod according to any of claims 4 to 12, in order to evaluate theefficiency or impact of treatments notably with agents having biologicalactivity, stresses, toxic agents, genotoxic aggressions.